Cell fractionator apparatus



March B4, 1967 c. J. FlLz ETAL CELL FRACTIONATOR APPARATUS 2Sheets-Sheet l Filed March 23, 1964 March 14, 1967 c. J. FlLz ETAL3,309,032

CELL FRACTIONATOR APPARATUS Filed Maron 25, 1964 2 sheets-sheet 2 INVENTORS United States Patent O 3,399,032 CELL FRACTIQNATGR APPARATUSCharles J. Filz, Weston, and Josef Blum, Norwalk, Coun.,

assignors to Ivan Sorvall, Inc., Norwalk, Conn., a corporation ofConnecticut Filed Mar. 23, 1964, Ser. No. 353,836 14 Claims. (Cl.241-301) This invention relates to an improved apparatus for rupturingcells in order to study the structure and function of their constituentparts.

For a long time it has been realized that research in the areaencompassing structure and function of microorganisms would be greatlyfacilitated if it were possible to rupture the cells and isolate thestructural elements such as cell walls and protoplasm withoutsignificant alteration of these components. Recently, a great deal ofattention has been directed toward development of tools and techniquesfor this purpose, and several instruments have been produced. Shakingand grinding devices, including the ultrasonic oscillator, have beenused to disrupt microorganisms but when separation of the disruptionproducts was attempted, a portion of the cell walls was fragmented tosuch an extent that it was impossible completely to remove them from theprotoplasm by differential centrifugation. Thus, the yields of pure cellwall material were small in proportion to the amount of startingmaterial, and protoplasm substantially free of cell Wall material couldnot be produced. Neither cell walls nor protoplasm could be isolated inappreciable amounts from Mycobacteria (tubercle bacilli) by the use ofthese 'devices due to the tendency of the broken and unbroken cells andthe protoplasm to aggregate to a butter-like consistency.

The French pressure cell, in which a pressurized microbial suspension isreleased at an orifice, seemed to be the most logical approach to theproblem, since pressures could be selected and regulated according tothe rigidity of the cell Walls of a given microorganism. The cells wouldcrack as they emerged through the oriiice and would not be subjected tofurther disintegration. When this method was used, recovery of cell wallmaterial was quite satisfactory, but temperatures at the oriiiceincreased to a point where partial denaturation of certain protoplasmicconstituents occurred, and this material adhered to the inner surface ofthe cell wall and could not be removed. Conventional cooling methodswere not effective.

It was then proposed that the orifice and needle valve of the pressurecell be exposed to a stream of pre-cooled gas that was released from aseparate needle valve. While this made it possible to produce somewhatbetter results in the form of purified cell walls and protoplasm, it wasnot until the introduction of the apparatus and method of the inventiondescribed and claimed in the copending U.S. patent application Ser. No.171,674, led Feb. 7, 1962, now U.S. Patent 3,165,266, issued Ian. 12,1965, that suitably controlled results in quantity could be obtained.

The present invention comprises an improvement over the inventiondescribed in said copending application in providing more eflicientmeans for cooling the valve seat and expansion or decompression chamberso that the yield of biological specimens suitable for analysis andexperimentation is considerably increased.

The improved cooling means comprises the inclusion of a plurality ofradially disposed channels surrounding the valve seat and the expansionchamber. The cooling medium is transmitted through said channels andexpelled into the expansion chamber in close proximity to the valveseat. By arranging for the channels to be radially arrayed around thevalve seat and around the expansion chamber, the block in which saidvalve seat and "ice chamber are formed is also cooled, whereby heatgenerated at the valve seat is effectively counteracted. More eicientcooling of the expansion chamber is realized by introducing the coolingmedium directly into the critical space of the chamber rather than bythe previous means of indirect circulation.

Still other objects and advantages of the invention will be apparentfrom the specification.

The features of novelty which are believed to be characteristic of theinvention are set forth herein and will best be understood, both as totheir fundamental principles and as to the particular embodiments, byreference to the specification and accompanying drawing, in which:

FIG. l is a vertical central section view of the improved fractionatirigvalve system and the outlet orifice cooling arrangement therefor, someparts being shown in partial elevation, and some parts being brokenaway;

FIG. 2 is a greatly enlarged section view of a portion 0f the apparatusshown in FIG. 1;

FIG. 3 is a further enlarged view of a portion of the apparatus, takenon line 3-3 of FIG. 2;

FIG. 4 is a view taken on line 4 4 of FIG. 2, partly in section andpartly in elevation; and

FIG. 5 is a schematic representation, in reduced size, but not inrelative scale, of the complete cell fractionating system of which thevalve apparatus shown in FIG. 1 forms an integral part.

Referring to the drawings, the cell fractionating process is shownschematically in FIG. 5, where a specimen 11 constituting such materialsas bacteria, fungi, spores, viruses, blood cells, and the like, inliquid suspension, is contained in a supply bottle 12. The specimen iscultured in the normal manner in nutrients which act as the liquidvehicle. Inserted into supply bottle 12 is one end of a flexible tube13, made of rubber, neoprene, or the like, the other end of which isconnected to the inlet port of high pressure one-way valve 14 which isadjusted by means of control rod 16, the outer end of which is providedwith a manually or mechanically operable knob 17.

Connected to the outlet port of valve 14 is one end of transfer tube 18,the other end of which is connected to the inlet port of pressure cell19. Pressure cell 19 has a cylinder 21 which slidably accommodates aplunger 22 connected to ram 23 mounted on piston 24 slidably positionedwithin hydraulic cylinder 26. Upon the outward motion of plunger 22 frompressure cell 19, suction is created whereby a specimen is drawn from aspecimen supply bottle 12 through valve 14 and into the cavity ofcylinder 21 of said cell. Upon the inward movement of plunger 22 intocylinder 21 pressure is applied to the specimen in the cavity of thepressure cell, causing the specimen to pass through transfer tube 28,one end of which communicates with cylinder '21, the other end of which-communicates with the interior of the expansion valve assembly,generally designate-d 29, the detailed structure of `which is shown inFIGS. 1, 2 and 3. Upon being processed through the expansion valve 29,the fractionated cells in the suspension medium pass through tube 31into a receiver bottle 32. Thereafter the materials in the receiverbottle may be transferred to a centrifuge where the components arephysically separated for such further study as may be required.

Tube 31 extends through a rubber stopper 33, or the like, in the mouthof the receiver bottle 32, said stopper also having a tube 34 extendingtherethrough to vent the cooling gas that had mixed with the eluent.

The control system for the hydraulic cylinder 26 comprises a sump orreservoir 36 of hydraulic liquid. A suction pipe line 37, extending intosump 36, is connected to a suitable pump 38 which causes the hydraulicfluid to pass by way of pipe line 39 through a three position valve 41,which is operated by a pair of solenoids 42 and 43.

J The solenoids are actuated by suitable -electric circuitry which iswell known in the art, and which does not constltute any part of thepresent invention. Valve 4I is shownY with the right hand section Apermitting free flow of hydraulic uid to and from hydraulic cylinder 26.The central section B of valve 41 is the no-go position, while thelefthand section C has a cross-over arrangement for reversing the ilowof the hydraulic fluid.

In;the position of the valve 41l shown in FIG. 5, hydraulic fluid thenpasses under pressure by way of pipe line 44 through ilter 46 and by wayof pipe line 47 into a flow control valve 48 which determines the rateflow of the hy-y draulic fluid through pipe line 49 into hydrauliccylinder 26.

It will be noted that pipe line 39 is provided with relief pipe line S1communicating with relief valve 52, the pressure control action of whichis represented schematically by spring 53, whereby excessive pressurecan berelieved by permitting llow of hydraulic liquid through outletpipe 54 into the sump 36. Flow control valve 48 is also provided with anoverow pipe line` 56 which also leads back to the sump 36.

The introduction of hydraulic fluid under pressure through pipe line 49kinto cylinder 26 is operative by way of piston 24, and ram 23 to driveplunger 22 into pressure cell cylinder 2.1.y This takes place when valve41 is in the position shown in FIG. 5. Overow of hydraulic fluid fromcylinder 26 during the pressure stroke returns through pipe lines 57-and 58 to sump 36. A retracting or suction stroke of plunger 22 isproduced by moving valve 41 to the right into the C position, wherebythe flow of hydraulic iluid is reversed and pipe line 57 becomes thepressure line, pipe line 49' becomes `the return-line, and pipe line 56is the overflow line into sump 36.- In the B position of valve 41, theaction of the hydraulic cylinder 26 is ata standstill, and if pump 38continues to operate, thehydraulic fluid simply lreturns to sump 36 byway of pipeline 58.

On the suction stroke of plunger 22, a specimen is'drawn from supplybottle into cylinder 21 of the pressure cell;

On the pressure stroke of plunger 22, the specimen is transmittedthrough pipe 28 to the expansion valve-29,

the return of the specimen to supply bottle 12 being pre- Y ventedy byone-Way valve 14.

A double-purpose cooling system is provided in order to maintainpressure cell 21 and expansion valve 29 kat the requisite lowtemperatures, since considerable heat is generated by the operation ofboth ofthese pieces'of apparatus. The cooling medium consisting of drynitrogen-air, or other neutral medium, is stored in supply tank 61 whichis provided with suitable pressure maintaining apparatus, not shown. Therefrigerating circuit, part in FIG. 5, consists of a cooling coil 62mounted in a suitable `heat exchanger 63. Coil 62 is supplied with acontinuous flow cooling medium, such as Freom'or the like, from arefrigerating sourcenot shown, through pipes `64 and 65.

The nitrogen is pumped through pipe 66 into cooling coil 67 in heatVexchanger 63y after whichthe cooled gas passes through pipek 68 to acoil 69 that extends around and cools pressure cell 19. Since thenitrogenabsorbs heat in coil 69, it is returned through pipe 71 to becooled again in coil 72 in heat exchanger 63. The re-cooled gas thenpasses ,through pipe 73 into the expansion valve assembly` 29 where itmaintains the requisite low temperature at the exit orifice of theruptured cells. Thencethe gas and the cells pass through tube 31 intothe receiver bottle 32, said gas then escaping into rthe. atmospherethrough vent tube 34.

As shown in FIGS. 1 3, the expansion valve assembly comprises a body76,'to the top of which is connected a nut 77 by means of a plurality ofbolts 78, and to the bottom of which -is connected a .bottom plate 79,by means of a plurality of bolts 81. Body 76,y nut 77, and bottom plate79 are made ofsteel or the like, and may be circular shown only in,A

in cross-section. In one 4embodiment their diameter is in i the order ofabout Ztl/z Nut 77 has a vertical central threaded aperture 82'` bore84, ,the upper end of which terminates in a threaded coaxial aperturev86 of somewhat greater diameter, which accommodates set screw 87 Bore84 in valve stem screw 83 movably accommodates an elongated valve stem.88, which also extends into bore 89 in body 76, bore 89 being coaxiallyaligned with bore 84. The lower end of valve stem 88 terminates in aconical valve head 91, whose function will be def scribed hereinafter.abuts the lower end of set screw 87 which can be adjusted to compensatefor wear onvalve head 91, land to maintain valve stern 88 in properposition in respect of stem screw 83. The` relative dimensions of valvestem 88 and of bore 84 of stem screw 83 provide a barely running t withextremely close clearance therebetween.

The lower end of bore 89 in body 76 terminates in a co-axial circularbore 92 of somewhat greater diameter. The lower end of bore 92communicates with a still larger co-axial, circular recess 93 whichaccommodates expansion valve seat bl-ock 94' 'which is maintainedcaptive therein by bottom plate 79. Interposed lbetween the perimeter ofblock 94 and the vertical walls of recess 93 is a circular. sleeve 95,made vof Teflon or other suitable insulating material, on top ofwhich isformed an inwardly extending integral collar 95a which mates with acorresponding recessed annular shoulder on top 1of said block.

Block 94 has an integral upwardly extending central tubular boss 96 thatts closely into bore 92. Bore 92 has an annular recess 97whichaccommodates resilient O-ring 98 rnade yof -a suitablematerial suchas.rubber, neoprene, Teflon or the like, which serves as a sealbetween/boss 96and body 76. .Positioned at the top of boss 96 isfO-ring99, also made of similar resilient material, whichy serves to seal valvestem 88 with adjacent parts. The sealing actioniof O-rings 98 and 99 isenhanced by the pressure conditions obtaining inthe system when theapparatus is in oper-ation as will'be described hereinafter.

The lower ,end of valve stem 88 extends co-axiallyy into the interior ofvalve chamber 101 in.tubular boss 96 and block 94, there being a slightclearance lbetween the surfaceyof, said stem andthe interior wall ofsaid chamber suflicient to permit liquid specimens to pass freelybetween said elements to ll valve ,chamber 101. Intermediate itsends,bossf 96 has a plurality of radially and equidistantly arrayedports 102 (FIGS. 2 and 3), the outer countersunk ends of whichcommunicate with anL annular recess 103 in body 76.y Recess103 serves todistribute specimen -materials substantially equally through ports 102into valve chamber 101. Distributor recess 103 communicates ,with ahorizontalspecimen transmitting channel104 which extends to threadedinlet recess 106 in body 76.

Since the specimen is delivered from cell 19 (FIG. 5) to the expansionvalve assembly 29 under high pressure, pipe line 28 is made of asubstantially rigidmaterial, which, inone embodiment, comprises a 1Ainch diameter pipehaving a 16th inch inside diameter. Pipe 28 has aground tapered end y107 which mates with a ground tapered seal 108 atthe inner end of recess y106 in body 76.

. In order to .ensure an intimate leak-proof tit 'between pipe end107and seat 108,1 the end portion of pipe 28 is threaded to accommodate athreaded ferrule 109. The annular wall of inlet recess 106' is threadedto accommodate threaded gland 111 whichr has a circular aperture 112into which ferrule 109 lits. Gland 111 has -an outwardly extendingintegral rim 113 to facilitate rotation thereofwhereby the interior wall114 of aperture 112 is The upper end of valve stern 88r forced againstthe end of ferrule 109 to cause tapered end 107 of pipe 28 to be urgedinto firm and intimate contact with seat 108. By this means a highpressure leak-proof connection is established between the interior ofpipe 28 and channel 104 in body 76.

Similar high pressure, leak-proof connections are provided between`pressure cell 19 `and pipes 18 and 28, respectively, and between pipe18 Iand high pressure unidirectional-valve 14. These high pressureconnections have a sufficient safety margin in strength to withstand thehigh pressure forces that are generated within pressure cell 19.

The lower end of transfer chamber 101 in block 94 has a conical orconcave conoidal constriction 116 which terminates in a small Circularneck 117 which, in turn, communicates coaxially with, and forms thesmaller end of, a conical open-mouthed expansion chamber 118. When valvestern screw S3 is turned to cause the lower portion of conical valvehead 91 to extend through neck 117, whereby an annular portion of valvehead 91 bears firmly against an annular portion of constriction 116which serves as a valve seat for said valve head, a leaktight seal isformed which withstands the high pressures generated by pressure cell19, thereby preventing the escape of the specimen from transfer chamber101 into expansion chamber 118.

The widened exit end of expansion chamber 118 communicates with theupper end of outlet nipple 121 extending through bottom plate 79.Connected to the lower end of nipple 121 is one end of tube 31 the otherend of which communicates with the interior of collection lbottle 32.

Extending partially into body 76 is a horizontal aperture 122 (FIGS. 1and 2.) which threadably accommodates an inlet nipple 123 (FIG. 5) towhich pipe 73 is connected. The inner end of aperture 122 communicateswith `an aperture 124 extending through an upper edge portion of sleeve95. The upper peripheral corner of block 94 is beveled to form anannular transfer channel 126 with which aperture 124 communicates.

Extending obliquely and radially through block 94 is a plurality ofcooling channels 127, the upper ends of which communicate with annulartransfer channel 126, the lower ends of said channels 127 communicatingin a circular array with expansion chamber 118 a short distance belowneck 117 of valve seat 116. See FIGS. 2 and 4.

In operation, the specimen materials in supply bottle 12 are drawn intocylinder 21 of pressure cell 19 by the retraction of plunger 22, withvalve 41 in the position where the C-portion thereof is arrayed betweenpipe lines 39-44 and 57-58. Thereafter, valve 41 is moved into the Aposition, as shown in FIG. 5, whereby the hydraulic system is operativeto cause plunger 22 to produce its pressure stroke in cylinder 21,thereby bringing the materials in said cylinder under high compression.One-way valve 14 prevents the specimen materials from returning intosupply bottle 12 while the forward end of the system including valvechamber 101 is closed by Valve head 91 bearing rrnly against valve seat116.

When the requisite pressure has been achieved upon the specimen in theclosed system, this is indicated by a suitable pressure gauge 131, orthe like, mounted in pipe line 49. Gauge 131 is calibrated Vto read thepressures in the closed specimen system by simple computation thatestablishes the relationship between the pressures imposed upon thelarge piston 24 in hydraulic cylinder 26, and the pressure translatedthereby through smaller plunger 22 in pressure cell 19.

Thereafter valve stem 88 is retracted very slightly by rotation of screw83 to barely back olf valve head 91l from constriction 116 by adistance, in one embodiment, in the order of about fifty-millionths ofan inch, to form an annular orice between valve head 91 and constriction116 through which the specimen escapes from chamber 101. U-pon passingthrough said orifice and through neck 117, the specimen is suddenlysubjected to explosive decompression in expansion chamber 118 andimmediate cooling by the cooling medium. Assisted by the flow .of thecooling gas, the exploded cells then move by gravity into and throughoutlet nipple 121, and thence through tube 31 into receiving bottle 32.

Since tremendous energy -is released in pressure cell 19 due to thecompression of the materials in cylinder 21, which, in one embodiment,reaches as much as 57,000 pounds per square inch, the cooling mediumfrom the refrigerating system is circulated around cell 19 through coil69 to absorb the heat generated and to chill the cell, and to keepcylinder 21 cool.

It is vitally important to keep the specimen cool as it undergoesdecompression; therefore, the cooling medium is conducted back into therefrigerating system through pipe 71 into heat exchange 63 forre-cooling, and thence it is passed by means of pipe 73 through inletnipple 123, aperture 122, aperture 124, annular transfer channel 126 anddown through cooling channels 127 whence it circulates into and through'expansion chamber 118. In this manner the neck 117 of valve seat 116 aswell as the space in the expansion or decompression chamber 118 aremaintained at a low temperature, which, in some operations, does notexceed approximately 5 C., or lower.

The foregoing means for introducing and circulating the cooling mediumclose to the juncture between the tip of valve head 91 and neck 117 ofvalve seat 116 where the critical energy conversion takes place,considerably increases the yield of useful biological specimens.

Not only is the critical temperature controlled at or Very close to thepoint of decompression and in .the area of expansion, but thearrangement for extending cooling channels 127 obliquely and radiallythrough block 94 also cools the latter whereby heat normally generatedby the fractionating process is absorbed and conducted away from thevalve seat which is an integral part of block 94.

Sleeve and collar 95a made of a suitable insulating material, serve asan insulator to reduce or prevent heat transfer between block 94 andbody 76. Said sleeve and collar also facilitate the assembly anddisassembly of block 94 relative to body 76, when cleaning andreplacement are necessary or desired.

By providing for a continuously circulating stream of the cooling mediumwithin the critical valve area, and within the decompression chamber,said medium mixing intimately with the fractionated cells as they emergefrom the valve chamber 101, it is possible, by means of the apparatusand system disclosed herein,vto preserve the fractionated cell particlesand constituents in a proper condition for further study, examination,analysis and experimentation.

The .intimately mixed fractionated cells and cooling medium passdownwardly through outlet nipple 124 and through pipe 31 into receivingbottle 32, where the effluents in liquid suspension are collected.

By providing for the conical valve head 91 to protrude throughconstriction 116 and through neck 117 where the materials under highpressure escape through an annular orifice, directly into a coaxialexpansion cham-ber, not only is the controlled retraction of valve head91 facilitated, but also it is now possible to ensure the controlledcooling of the critical area in which the specimens under high pressureundergo explosive decompression or expansion. This was hithertoincapable of being achieved where the specimen under pressure Howed in adirection opposite to the valve head, thereby preventing access of acooling medium to the valve head and the valve seat, in order topreserve the integrity of the specimen structures. Also, by providingfor specimen flow in the same direction as the Valve head, .into acoaxial expansion chamber, the specimen does not impinge upon anyobstruction `to its flow which would otherwise denaturize thefractionated material.

The arrangement of making the valve stem structure in two parts, namely,sternk 8S and screw 83, constitutes an improvementover aone'partstructure. Due to the precision requirements of providing thecontrolled minute orice between valve head 91 and constriction ,116,withaccurate concentricity of the conical end of said valve head in respectof valve'seat and of neck 117 for even distribution of the specimen, ithas been found that valve stem 88 and its head 91 can more readily beaccurately machined las a part separate from valve stem screw 83, whosebore 84 can also -be more accurately machined in relation to'bore 89fin'body 76.. The relative positions of valve stem 88 andvalve screw 83 toeach other are established by set screw S7.

After the requisite pressure of the specimen has been achieved in thesystem, valve head 91 is very slightly retracted from constriction 116by rotating valve stem screw 83 asmall fraction of a turn. While vthespecimen is escaping through neck 117, the pressures wit-hin the systemare suicient to cause the specimen in valve chamber 101 to` bear uponvalve head 91 and to urge valve stem 88 upwardly against the lower endof set screw 87, whereby the requisite position of said valve stem 88relative to the position of valve stemy screw S3 is maintained. Leakageof specimen fluids from between the valve chamber 16'1 and body 76 isprevented by O-rings 98 and 99, the internal pressure of the system inactuality enhancing the sealing action of said O-rings.

Should any wear occur on either or both valve head 91 and constriction116fdue to frictional engagement therebetween, the position of valvestem 88- may be relocated in respect of valve stem screw 83 by adjustingset screw S7. Since the valve stem, valve head, and valve seat areaccurately machined, any wear that takes place therebetween issubstantiallyuniform, and, therefore, a re-setting of set screw 83 issufficient to maintain the apparatus in working order.

The complete system, shown schematically in FIG.'5, may be enclosed in asuitable cabinet, or the like, not shown. In some embodiments it may bedesirable to arrange for some of the components to be compartmented intoa separate enclosure, indicated by thedotted lines 132, so that theprocessingof the specimens may take place in an environment which maybeirradiated by ultravoilet or perfused with various materials, such asinert gases or bactericidal or germicidal sprays in order to protect thespecimens from contamination. Furthermore, enclosure 132 served toprotect the operator when certain types of infectious or otherwisedangerous materials are being processed by the'k apparatus.

Suitable leak-tight connectionsV are provided in compartment 132,whereby the cooling medium may be introduced and withdrawn, theoperation of the pressurey cell piston facilitated by a flexibleair-tight juncture 1153 and for various external controls for settingthe pressure value of one-way valve 14, and operating valve stem screw83, andthe like. Furthermore, such leak-tight connections serve not onlyto prevent contamination of the external controls and apparatus, butalso serve to prevent the escape of contaminating materials into theatmosphere of the laboratory.

It is clai-med:

1. Cell fractionating apparatus comprising a block, a compressioncham-ber in said block, and expansion chamber in said block axiallyaligned with said cornpression chamber, a valve seat between saidcompression chamber and said expansion chamber, a valve member movablelongitudinally within said compression chamber and cooperating with saidvalve seat, the entry, portion of said expansion chamber being conicalin shape and the apex thereof terminating in said valve seat, ymeans forintroducingbiological specimens under high pressure into saidcompression chamber, a plurality of radially arrayed channels in said|block, the inner ends of said channels communicating in a circulararray with the conical portion of said expansion chamber near said valveseat and means for passing a cooling medium simultaneously through allof said channels into said expansion chamber.

2. Apparatus according to claim 1 wherein said channels are arrayedobliquely within said block, the inlet ends of said channels beinglocated in the upper portion of said block whereby said coolingmediuinis passed downwardlyV and inwardly toward said expansion chamber.

3. Cell fractionating apparatus comprising a block, a compressionchamber in said block, an expansion chamber in said block axiallyaligned with said compression chamber, a constriction betweensaidcompression chamber and said expansion ychamber forming ,a valve seat, avalve member movable longitudinally within said compression chamber andcooperating kwith said valve seat, a plurality of channelsin said blockradially arrayed around and ycommunicating with the entry portion ofsaid expansion chamber, the outlets of said channelsl forming a circulararray near said. valve seat, means for introducing biological specimensunder 'pressurey intoI said compression chamber, and meansfortransmitting a cooling medium through said channels, said cooling meansentering said expansion chamber near said valve seat.

4. Apparatus according to claim 3 wherein said valve prising anannularchannelin said block, the outer endsl of said radial `channelscommunicating with said annular channel,`said annular channeltransferring said cooling medium from said transmitting meanssimultaneously to said radial channels.

6. Apparatus according to, claim 3 and further comprising a body, saidintroducing means and said transmitting means being located in saidbody, a recess in said body accommodating said block and `an insulatingsheath intermediate said block land said body.

7. Apparatus according to claim 3 andfurther comprising a body, saidintroducing means Iand `said transmitting means being located in said`body, a recess in said body accommodating said block, an insulatingsheath .intermediate said block and said body, an annular channel insaid Afblock, the outer ends of said radial channels comf' municatingwith said annularchannel, an aperture lin said sheath, said apertureestablishing communication between said transmitting means and said'annular -channel for transferring said cooling medium to saidradialchannels.

8. Cellfractionating apparatus comprising a block,: a compressionchamber in said block, an expansion chamber in said block, a valve`between said chambers, and a plurality of channels in said block, theoutlets of said channels being located in a circular array around andcommunicating withthe entry portion of said expansion chamber near saidvalve.

9. Cell fractionating apparatus comprising a block, a compressioncham-ber in said block, an expansion chamber in said block axiallyaligned with said compression chamber, a valve between said chambers,the entry portion ofsaid expansion chamber being conical in shape andthe apex thereof terminating in said valve, means for introducingbiological specimens into said compression chamber, and means forintroducing and circulating cooling medium into `said entry portion ofsaid expansion chamber close to said valve.

10. Cell fractionating apparatus comprising a block, a compressionchamberr in said block, an expansion chamber in said block Vvaxiallyaligned withsaid compression chamber, a valve between said chambers, theentry portion of said expansion chamber being conical in shape and theapex thereof terminating in said valve, means for introducing biologicalspecimens under pressure into said compression chamber, and means fortransmitting a cooling medium through said block and into said entryportion of said expansion chamber to cool the latter and to increase theheat absorption of said block due to decompression of said specimens.

11. Cell fractionating apparatus comprising a block, a compressionchamber in said block, an expansion chamber in said block, rst means forcontrolling the passage of materials from said compression chamber intosaid expansion chamber, and second means for cooling said block and theentry portion of said expansion chamber near said first means.

12. Apparatus according to claim 11 wherein said rst means comprises anormally closed valve between said compression chamber and saidexpansion chamber.

References Cited by the Examiner UNITED STATES PATENTS 2,021,143 11/1935Calcott 252-6 2,841,384 7/1958 Petersen 263-32 2,893,216 7/1959 Seefeldtet al. 62-63 3,165,266 1/1965 Blum et al. 241-1 3,172,546 3/1965Schreiner 241-23 3,206,755 9/1965 Friedman 346-1 WILLIAM W. DYER, IR.,Primary Examiner. R. ZLOTNIK, Assistant Examiner.

1. CELL FRACTIONTING APPARATUS COMPRISING A BLOCK, A COMPRESSION CHAMBERIN SAID BLOCK, AND EXPANSION CHAMBER IN SAID BLOCK AXIALLY ALIGNED WITHSAID COMPRESSION CHAMBER, A VALVE SEAT BETWEEN SAID COMPRESSION CHAMBERAND SAID EXPANSION CHAMBER, A VALVE MEMBER MOVABLE LONGITUDINALLY WITHINSAID COMPRESSION CHAMBER AND COOPERATING WITH SAID VALVE SEAT, THE ENTRYPORTION OF SAID EXPANSION CHAMBER BEING CONICAL IN SHAPE AND THE APEXTHEREOF TERMINATING IN SAID VALVE SEAT, MEANS FOR INTRODUCING BIOLOGICALSPECIMENS UNDER HIGH PRESSURE INTO SAID COMPRESSION CHAMBER, A PLURALITYOF RADIALLY ARRAYED CHANNELS IN SAID BLOCK, THE INNER ENDS OF SAIDCHANNELS COMMUNICATING IN A CIRCULAR ARRAY WITH THE CONICAL PORTION OFSAID EXPANSION CHAMBER NEAR SAID VALVE SEAT AND MEANS FOR PASSING ACOOLING MEDIUM SIMULTANEOUSLY THROUGH ALL OF SAID CHANNELS INTO SAIDEXPANSION CHAMBER.